- Email: [email protected]
Intracellular localization of β-aspartate kinase in spinach (spinacea oleracea)
AlFgust 1979
lq htraduction The aspartate-derived amino acid pathway leads to the synthesis of lysine, threonine and methionine, essential in the nutrition of nonruminant animals, including man. The location of the b~~s~~etic MacWeek for this pathway in the c~oro~lasts of hi@%erplants is indicated by the dem~~strati~ of the activity of: ~~~~tate kinase in pea ~~oroplasts and root plastids (Burdge et al., in preparation), a iysineand thraonine-sensitive j3-aspartate kinsss from pea leaf chloroplasts isolated by differentillr. ceatrifugation [I I3 diaminopimelate decarboxylasa from I/i&r faba [2J1and homoserine dehydrogenase from maize [3X,Isolated pea chloroplasts are also capable of lightdependent incorporation of radiolabeled sspartate and sulfate into soluble and protein-bold amino acids of the asp&ate family @+if_ These bogs sug&st the irn~~~~ of the chloroplast in nitrogen met~bo~sm. P-Aspartate kinase is a key regulatory enzyme catalyzing the first step in the aspartate pathway leading to the synthesis of lysine, thteoaine and methionine. As such, its presence in s perticular cellular compartment or organelle would suggest that control of the aspartate pathway resides:in that [email protected] present work reports resuits of initial efforts to demonstrate the presence of j%aspartate
lcinase (ATP: L*sspartate 4 phosphotransferass, EC 2.7.2.4) in organelle preparations from spinach leaf tissues.
Deribbed tissue of spinach (cv. ~~oo~sd~~ Longstanding) leaves from one-month-old plants grown at 22°C with 250 ps vMq2 mixed incandescent and fluorescent l&ht for 8 h daily was ground in a 66 mM Tricine buffer @I 8,4,1:4 w/v) containing 0.33 M sorbitol, 2 mM M&la, 1 mM EDTA, 4 mM 2-mercaptoethanol, and 0.1% bovine serum albumin, A SorvaB omni mixer was used in sucrose gradient prepsration~~ and a polytron l~omo~~~er was employed for differential ce~t~f~~tion= The crude homoge~te was filtered through several layers of cheesecloth and miracloth.
A combiued continuous and discontinuous sucrose gradient was used to isolate intact chloroplasts according to Miflin and Beevers [5] (O-&C). The crude homogenate was centrifuged at 1500 X g for 1 min. The resulting pellet was resuspended in extraction buffer minus mer~~t~~ol and centrifuged at 1500 X g for 1 min. This @let was ~sus~~ded in emaction buffer minus rne~~t~~ol~ and 10 ml of the resuspended pellet were layered on top of the sucrose gradient, consisting of a 4-ml cushion of 60% 303
Volume 104, number 2
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sucrose, a 6-ml linear gradient of 60-42% sucrose, 5 ml of 42% sucrose, a lo-ml linear gradient of 42-30% sucrose, and a final 3 ml of 30% sucrose. After addition of the homogenate, the gradients were placed in a SW-27 rotor in a Beckman L.5-65 centrifuge and centrifuged at 2500 X g for 5 min and then 18 000 X g for an additional 10 min. The gradients were fractionated into 1.5-ml samples using an ISCO Model 328 density gradient fractionator. Sucrose concentrations were determined by refractometry. Protein concentrations for each fraction were determined, following protein precipitation with 10% TCA, by a microbiuret assay, using bovine serum albumin (Sigma) as a standard [6]. Chlorophyll was determined on each fraction by the method of Arnon 171. 2.3. Chloroplast preparation by differential ten tifigation The spinachleaf crude homogenate was centrifuged
at 2000 X g for 50 s (O&C). The resulting pellet (Pr ) was resuspended in the exttaction buffer and centrifuged twice at 2000 X g for 50 s. The resulting washed pellet (Ps) was then resuspended in extraction buffer + 25% glycerol and sonicated (Ultrasonics, Inc.) with 2-3-s bursts at 60 W power. This solution was then centrifuged at 2000 X g for 50 s to obtain a supematant (FPS) and the pellet (FPP). The pellet (FPP) was resuspended in extraction buffer t 25% glycerol. The crude, supernatant and pellet fractions were analyzed for protein (by a modified Lowry [B] ), chlorophyll, fl-aspartate kinase and nitrite reductase activities. This pellet (Pa) was also analyzed for chloroplast integrity by ferricyanide-dependent O2 evolution and CO? fmation. 2.4. Fem’cyanidedependen t O2 evolution The ferricyanide reaction was used as a criterion for intactness of the chloroplast envelope [9]. The degree of intactness of the chloroplasts was estimated from rates of ferricyanide-dependent O2 evolution before and after osmotic rupture. The oxygen concentration was measured in a YSI model 5331 Oxygen Monitor in a solution containing 50 mM HEPES (pH 7.6), 0.33 M sorbitol, 2 mM EDTA, 1 mM MgCls and 1 mM Mm&. Six ~1 of KaFe(CN)e (0.1 M), 18 fi of 0.1 M NH.+Cl,and chloroplast suspension corresponding to -10 c(g of chlorophyll were 304
August 1979
added to a final volume of 2 ml. Oxygen evolution from ruptured chloroplasts was measured in the same HEPES buffer minus sorbitol. 2.5. CO2 fDcation Light-dependent CO? furation was measured according to Walker [lo]. The following reagents plus NaHr4C0s (243 dpm/nmole, New England Nuclear) and 50 pg/ml chlorophyll were added to a final volume of 2 ml: 50 mM HEPES @H 7.6, adjusted with NaOH), 0.33 M sorbitol, 2 mM EDTA, 1 mM MgC12,1 mM Mm&, 2 mM Naisoascorbate, 0.5 mM K,HP04, and 5.0 mM Na4PZ0,. At 2-min intervals for 10 min, 200 r_llof the assay mixture were removed and acidified with 160 /..dof 5 N HCl. The samples were oven-dried, and r4C02 fmation, measured as acid-stable product (in 2: 1 toluene:Triton containing PPO and POPOP), was determined in a Mark.111 Searle scintillation counter. Activity is reported as nmol COZ fixed/mg chl - h. 2.6. Enzyme assays All enzymes were measured spectrophotometrically at 25°C using a Beckman Model 25 spectrophotometer. The following marker enzymes were assayed on each sucrose density.gradient fraction: catalase (EC 1.11 .1.6) [ll], cytochrome c oxidase (EC 1.9.3.1) [12], and triosephosphate isomerase (EC 5.2.1 .l) [13]. Nitrite reductase [ 141 and P-aspartate kinase by the method of Black and Wright [ 151 as previously described [ 161 were also assayed.
3. Results and discussion The distribution of /?-aspartate kinase activity in the sucrose density gradients indicates that a sharp peak of fl-aspartate kinase coincides with the chlorophyll and triosephosphate isomerase activity peaks in the intact chloroplast (fraction no. 7) (fig.lA-C). This chloroplast peak is essentially free of cytochrome c oxidase and catalase activities, and thus mitochondria and microbodies. A substantial portion of the P-aspartate kinase activity occurs in the region of the gradient corresponding to the major chlorophyllcontaining fractions (fraction no. 10-20). These fractions represent damaged chloroplasts which are devoid of stroma. In addition, a substantial portion of
FEBS LETTERS
Volume 104, number 2
the @-aspartatekinase activity appears to coincide with the cytosolic form of triosephosphate isomerase, at the top of the gradient. The pattern of distribution obtained for /I-aspartate kinase suggests that a chloroplast- or thylakoid-localized form of /I-aspartate kinase is present, or that /3-aspartate kinase binds tightly to the outer membrane of the chloroplast. Representation of these data in tabular form. (table 1) substantiates the apparent enrichment of
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/I-aspartate kinase in the intact chloroplast fractions, although it does seem to be distributed also in the soluble and broken chloroplast fractions. The activity associated with these fractions may have resulted from damage to the chloroplasts by the fractionation procedures; however, there is approximately 30% enrichment of the /I-aspartate kinase activity in the intact chloroplast fraction compared to the other fractions. The appearance of /3-aspartatekinase activity in the chloroplast peak suggests that /3-aspartate kinase is partially localized within the chloroplast. To further substantiate the localization of /3-aspartate kinase within the chloroplasts, a second method (described in 2.3) was employed to isolate intact chloroplasts. Results of a representative differential centrifugation are shown in table 2. The distribution of nitrite reductase and the rate of CO? fixation indicate that a substantial portion of the chloroplasts have leaked their contents. The chloroplast pellet contained approximately 40-50% intact chloroplasts as determined by ferricyanide-dependent O2 evolution and fmed low rates of COa - 10 ~01 COJmg chl . h; the ferricyanide assay, however, does not distinguish between intact chloroplasts and those that have opened and resealed. (Subsequent experiments have yielded chloroplast preparations which are approximately 70% intact.) Six to seven percent of the total nitrite reductase activity was found in the chloroplast pellet. Assuming the pellet contains approximately 50%intact chloroplasts, approximately 12-14% of the nitrite reductase total activity can be attributed to the chloroplast. This finding agrees with that of h4iflin; i.e., approximately 13% of the nitrite reductase is associated with the intact chloroplast fraction from sucrose density gradient preparations [ 171, which indicates substantial breakage of the chloroplasts inherent in grinding and centrifugation procedures. P-Aspartate kinase also shows a substantial portion of activity in the supernatant, with approximately 11% associated with the chloroplastenriched pellet. If the chloroplasts were 40-50% intact, the most activity that could be attributed to the chloroplast fraction would be approximately 20-25% of the total, roughly equivalent to what was observed in the sucrose density gradient intact chloroplast peak. However, quantitative determination of the estimates of in vivo enzyme content in the chloroplasts by a
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Fig.lA-C. Results of sucrose density gradient centrifugation of young, deribbed spinach leaves.
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Volume 104, number 2
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August 1979
Table 1 Enzyme and chlorophyll distribution in sucrose density gradient fractions Component
Intact chloroplasts
Mitochondria and broken chloroplasts
Microbodies and soluble fraction
Chlorophyll &ml % of total Catalasea % of total Triosepho~hate isomerasea % of total Cytochrome oxidasea % of total p-Aspartate kinaseb % of total
1296.3 (48) 56.3 (9.91 3.92 (4.6) 0.411 (18) 4.44 (39)
894.2 (40) 81.3 (14) 4.41 (5.2) 1.586 (70) 3.04 (27)
140.1 (5.7) 409.2 (72) 65.66 (78) 0.084 (3.7) 3.22 (28)
a fimol/ml . mm b pmol/ml . h
calculation of recovered enzyme activity in relation to recovered chlorophyll in the intact chloroplast, used by Bryan et al. [3] for homoserine dehydrogenase, indicates that appro~m~ely 60% of the total &aspartate kinase activity is associated with the intact chloroplast . Analysis of the FPS and FPP fractions shows that the only detectable nitrite reductase activity was associated with the soluble fraction and that the
distribution of P-aspartate kinase is roughly equivalent in the two fractions; therefore, we do not yet know if &aspartate kinase is a soluble stromal or membranebound protein in the c~oropl~t. The data suggest that either two populations exist or fi-aspartate kinase sticks to the membrane. The intracellular distribution of fl-aspartate kinase in a leaf homogenate was also determined by another procedure, This involved centrifugation of a crude
Table 2 Distribution of nitrite reductase and @-aspartate kinase activities among differential centrifugation fractions Fraction
Crude Super, Super, Super 3 Pelletd 56 of pellet FPS FPP
CNorophyU
Protein
Nitrite reductase
@-Aspartate kinase
&ml
% total
mg/ml
% total
Total activitya
% total
Total activity b
% total
45 10 7.6 13 166
100 20 1.5 2.7 56
1.7 1.2 0.6 0.5 2.3
100 62 3 2.1 20
35.8 22.4 NDC ND 2.39
100 62 6.7
310 258 21.8 4.6 34.3
100 83 7.0 1.4 11
67 90
43 57
1.2 1.6
42 58
0.57 ND
26
a Units ‘=mmol NO; red/mm b Units = rrnoi AspHjmin ’ ND = not detectable dCharacteristics of chloroplasts -50% intact, fii 10 rmol CO,/mg chl * h
306
12.3 13.6
36 40
August 1979
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Volume 104, number 2
Table 3 Solubilization of p-aspartate kinase from isolated chloroplasts Specific activitya (units/mg protein)
+BSA (O.l%)b
Chlorophyll
-BSAb
% recovered
Total units
% total
% protein
Total units
% total
% protein
100
100
1142
100
100
832
70
71
5
13
Crude suspension Supernatant solution minus whole
2.17
100
2490
chloroplasts Extract from ruptured chloroplasts Supernatant/peUet enzyme ratio
1.85
42
1296
52
46
3.06
62
95
4
6
18.84
56.7 14.67
Under standard assay conditions, 60 min, 30°C, a and b are representative samples of separate experiments
homogenate at 500 X g for 1 min to sediment whole cells and debris. The resulting supernatant was centrifuged at 1500 X g for 5 min to sediment whole chloroplasts. Analysis of these fractions for /I-aspartate kinase activity yielded the following results (table 3). /3-Aspartatekinase specific activity in units/mg protein increased from 2.17 to 3.06 in the ruptured chloroplast fractions and was therefore enriched in the chloroplast pellet. The enrichment of the fl-aspartate kinase activity in the chloroplast pellet may be due to nonspecific binding of the protein to the membrane sites on the chloroplasts. To determine if P-aspartate kinase was binding nonspecifically, 0.1% bovine serum albumin was added to the extraction buffer, with the following results (table 3). /3-Aspartatekinase was approximately 5% of the crude preparation in both plus and minus bovine serum albumin preparations, showing no difference in the distribution of the enzyme activity. The supematant/pellet ratios were also approximately similar. The addition of bovine serum albumin to the extraction buffer also seemed to protect the enzyme activity, as noted in the increase in the total percentage recovered. Although some non-specific binding of /J-aspartate kinase may occur, the distribution of /.I-aspartate kinase in sucrose gradients and differential centrifugation indicates that there is a chloroplast-localized /3-aspartate kinase. Experiments are underway to characterize the chloroplast-localized P-aspartate kinase and the cytosolic /3-aspartate kinase, and to
determine if these are the same enzyme or isoenzyme forms by studying biochemical, kinetic and regulatory properties of the enzyme(s).
Acknowledgements This work was supported by the University of Tennessee Institute of Agriculture and US Department of Energy Contract no. EY-76-C-054242 with the University of Tennessee. The authors gratefully acknowledge the expert technical advice, critical review of the manuscript and use of equipment (oxygen monitor) provided by Dr Frederick J. Ryan and the critical review of the manuscript provided by Dr Otto J. Schwarz.
References [l] Lea, P. J., Mills, W. R. and Miflin, B. J. (1979) FEBS Lett. 98,165-168. [2] Mazehs, M., Miflin, B. J. and Pratt, H. M. (1976) FEBS Lett. 64,197-200. [3] Bryan, J. K., Lisik, E. A. and Matthews, B. F. (1977) Plant Physiol. 59,673-679. (41 Mills, W. R. and Wilson, K. G. (1978) FEBS Lett. 92, 129-132. [S] Miflin, B. J. and Beevers, H. (1974) Plant Physiol. 53, 870-874. [6] Itzaki, R. F. and Gill, D. M. (1964) Anal. Biochem. 9, 401-410. [7] Arnon, D. I. (1949) Plant Physiol. 24,1-15.
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[8] Schacterle, G. R. and Pollack, R. L. (1973) Anal. Biochem. 51,654-655. [9] Lilley, R. McC., Fitzgerald, M. P., Rienits, K. G. and Walker, D. A. (1975) New Phytol. 75, l-10. [lo] Walker, D. A. (1971) Methods Enzymol. 23,211-220. [ 1 l] Luck, H. (1965) in: Methods in Enzymatic Analysis (Bergmeyer, H.-U. ed) pp. 885-894, Academic Press, New York. [12] Hackett, D. P. (1964) in: Modern Methods of Plant Analysis (Linskins, H. F., Sanwal, B. D. and Tracey, M. V. eds) vol. VII, p. 647, Springer-Verlag. New York.
308
August 1979
[13] Gibbs, M. and Turner, J. F. (1964) in: Modem Methods of Plant Analysis (Linskins, H. F., Sanwal, B. D. and Tracey, M. V. eds) vol. VII, p. 520, Springer-Verlag, New York. (141 Losada, M. and Paneque, A. (1971) Methods Enzymol. 23,487-491. [15] Black, S. and Wright, N. G. (1955) J. Biol. Chem. 213, 27-38. [ 161 Henke, R. R. and Wahnbaeck, R. (1977) Biochem. Biophys. Res. Comm. 79,38-45. [ 171 Mifhn, B. J. (1974) Plant Physiol. 54,550-555.
lq htraduction The aspartate-derived amino acid pathway leads to the synthesis of lysine, threonine and methionine, essential in the nutrition of nonruminant animals, including man. The location of the b~~s~~etic MacWeek for this pathway in the c~oro~lasts of hi@%erplants is indicated by the dem~~strati~ of the activity of: ~~~~tate kinase in pea ~~oroplasts and root plastids (Burdge et al., in preparation), a iysineand thraonine-sensitive j3-aspartate kinsss from pea leaf chloroplasts isolated by differentillr. ceatrifugation [I I3 diaminopimelate decarboxylasa from I/i&r faba [2J1and homoserine dehydrogenase from maize [3X,Isolated pea chloroplasts are also capable of lightdependent incorporation of radiolabeled sspartate and sulfate into soluble and protein-bold amino acids of the asp&ate family @+if_ These bogs sug&st the irn~~~~ of the chloroplast in nitrogen met~bo~sm. P-Aspartate kinase is a key regulatory enzyme catalyzing the first step in the aspartate pathway leading to the synthesis of lysine, thteoaine and methionine. As such, its presence in s perticular cellular compartment or organelle would suggest that control of the aspartate pathway resides:in that [email protected] present work reports resuits of initial efforts to demonstrate the presence of j%aspartate
lcinase (ATP: L*sspartate 4 phosphotransferass, EC 2.7.2.4) in organelle preparations from spinach leaf tissues.
Deribbed tissue of spinach (cv. ~~oo~sd~~ Longstanding) leaves from one-month-old plants grown at 22°C with 250 ps vMq2 mixed incandescent and fluorescent l&ht for 8 h daily was ground in a 66 mM Tricine buffer @I 8,4,1:4 w/v) containing 0.33 M sorbitol, 2 mM M&la, 1 mM EDTA, 4 mM 2-mercaptoethanol, and 0.1% bovine serum albumin, A SorvaB omni mixer was used in sucrose gradient prepsration~~ and a polytron l~omo~~~er was employed for differential ce~t~f~~tion= The crude homoge~te was filtered through several layers of cheesecloth and miracloth.
A combiued continuous and discontinuous sucrose gradient was used to isolate intact chloroplasts according to Miflin and Beevers [5] (O-&C). The crude homogenate was centrifuged at 1500 X g for 1 min. The resulting pellet was resuspended in extraction buffer minus mer~~t~~ol and centrifuged at 1500 X g for 1 min. This @let was ~sus~~ded in emaction buffer minus rne~~t~~ol~ and 10 ml of the resuspended pellet were layered on top of the sucrose gradient, consisting of a 4-ml cushion of 60% 303
Volume 104, number 2
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sucrose, a 6-ml linear gradient of 60-42% sucrose, 5 ml of 42% sucrose, a lo-ml linear gradient of 42-30% sucrose, and a final 3 ml of 30% sucrose. After addition of the homogenate, the gradients were placed in a SW-27 rotor in a Beckman L.5-65 centrifuge and centrifuged at 2500 X g for 5 min and then 18 000 X g for an additional 10 min. The gradients were fractionated into 1.5-ml samples using an ISCO Model 328 density gradient fractionator. Sucrose concentrations were determined by refractometry. Protein concentrations for each fraction were determined, following protein precipitation with 10% TCA, by a microbiuret assay, using bovine serum albumin (Sigma) as a standard [6]. Chlorophyll was determined on each fraction by the method of Arnon 171. 2.3. Chloroplast preparation by differential ten tifigation The spinachleaf crude homogenate was centrifuged
at 2000 X g for 50 s (O&C). The resulting pellet (Pr ) was resuspended in the exttaction buffer and centrifuged twice at 2000 X g for 50 s. The resulting washed pellet (Ps) was then resuspended in extraction buffer + 25% glycerol and sonicated (Ultrasonics, Inc.) with 2-3-s bursts at 60 W power. This solution was then centrifuged at 2000 X g for 50 s to obtain a supematant (FPS) and the pellet (FPP). The pellet (FPP) was resuspended in extraction buffer t 25% glycerol. The crude, supernatant and pellet fractions were analyzed for protein (by a modified Lowry [B] ), chlorophyll, fl-aspartate kinase and nitrite reductase activities. This pellet (Pa) was also analyzed for chloroplast integrity by ferricyanide-dependent O2 evolution and CO? fmation. 2.4. Fem’cyanidedependen t O2 evolution The ferricyanide reaction was used as a criterion for intactness of the chloroplast envelope [9]. The degree of intactness of the chloroplasts was estimated from rates of ferricyanide-dependent O2 evolution before and after osmotic rupture. The oxygen concentration was measured in a YSI model 5331 Oxygen Monitor in a solution containing 50 mM HEPES (pH 7.6), 0.33 M sorbitol, 2 mM EDTA, 1 mM MgCls and 1 mM Mm&. Six ~1 of KaFe(CN)e (0.1 M), 18 fi of 0.1 M NH.+Cl,and chloroplast suspension corresponding to -10 c(g of chlorophyll were 304
August 1979
added to a final volume of 2 ml. Oxygen evolution from ruptured chloroplasts was measured in the same HEPES buffer minus sorbitol. 2.5. CO2 fDcation Light-dependent CO? furation was measured according to Walker [lo]. The following reagents plus NaHr4C0s (243 dpm/nmole, New England Nuclear) and 50 pg/ml chlorophyll were added to a final volume of 2 ml: 50 mM HEPES @H 7.6, adjusted with NaOH), 0.33 M sorbitol, 2 mM EDTA, 1 mM MgC12,1 mM Mm&, 2 mM Naisoascorbate, 0.5 mM K,HP04, and 5.0 mM Na4PZ0,. At 2-min intervals for 10 min, 200 r_llof the assay mixture were removed and acidified with 160 /..dof 5 N HCl. The samples were oven-dried, and r4C02 fmation, measured as acid-stable product (in 2: 1 toluene:Triton containing PPO and POPOP), was determined in a Mark.111 Searle scintillation counter. Activity is reported as nmol COZ fixed/mg chl - h. 2.6. Enzyme assays All enzymes were measured spectrophotometrically at 25°C using a Beckman Model 25 spectrophotometer. The following marker enzymes were assayed on each sucrose density.gradient fraction: catalase (EC 1.11 .1.6) [ll], cytochrome c oxidase (EC 1.9.3.1) [12], and triosephosphate isomerase (EC 5.2.1 .l) [13]. Nitrite reductase [ 141 and P-aspartate kinase by the method of Black and Wright [ 151 as previously described [ 161 were also assayed.
3. Results and discussion The distribution of /?-aspartate kinase activity in the sucrose density gradients indicates that a sharp peak of fl-aspartate kinase coincides with the chlorophyll and triosephosphate isomerase activity peaks in the intact chloroplast (fraction no. 7) (fig.lA-C). This chloroplast peak is essentially free of cytochrome c oxidase and catalase activities, and thus mitochondria and microbodies. A substantial portion of the P-aspartate kinase activity occurs in the region of the gradient corresponding to the major chlorophyllcontaining fractions (fraction no. 10-20). These fractions represent damaged chloroplasts which are devoid of stroma. In addition, a substantial portion of
FEBS LETTERS
Volume 104, number 2
the @-aspartatekinase activity appears to coincide with the cytosolic form of triosephosphate isomerase, at the top of the gradient. The pattern of distribution obtained for /I-aspartate kinase suggests that a chloroplast- or thylakoid-localized form of /I-aspartate kinase is present, or that /3-aspartate kinase binds tightly to the outer membrane of the chloroplast. Representation of these data in tabular form. (table 1) substantiates the apparent enrichment of
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/I-aspartate kinase in the intact chloroplast fractions, although it does seem to be distributed also in the soluble and broken chloroplast fractions. The activity associated with these fractions may have resulted from damage to the chloroplasts by the fractionation procedures; however, there is approximately 30% enrichment of the /I-aspartate kinase activity in the intact chloroplast fraction compared to the other fractions. The appearance of /3-aspartatekinase activity in the chloroplast peak suggests that /3-aspartate kinase is partially localized within the chloroplast. To further substantiate the localization of /3-aspartate kinase within the chloroplasts, a second method (described in 2.3) was employed to isolate intact chloroplasts. Results of a representative differential centrifugation are shown in table 2. The distribution of nitrite reductase and the rate of CO? fixation indicate that a substantial portion of the chloroplasts have leaked their contents. The chloroplast pellet contained approximately 40-50% intact chloroplasts as determined by ferricyanide-dependent O2 evolution and fmed low rates of COa - 10 ~01 COJmg chl . h; the ferricyanide assay, however, does not distinguish between intact chloroplasts and those that have opened and resealed. (Subsequent experiments have yielded chloroplast preparations which are approximately 70% intact.) Six to seven percent of the total nitrite reductase activity was found in the chloroplast pellet. Assuming the pellet contains approximately 50%intact chloroplasts, approximately 12-14% of the nitrite reductase total activity can be attributed to the chloroplast. This finding agrees with that of h4iflin; i.e., approximately 13% of the nitrite reductase is associated with the intact chloroplast fraction from sucrose density gradient preparations [ 171, which indicates substantial breakage of the chloroplasts inherent in grinding and centrifugation procedures. P-Aspartate kinase also shows a substantial portion of activity in the supernatant, with approximately 11% associated with the chloroplastenriched pellet. If the chloroplasts were 40-50% intact, the most activity that could be attributed to the chloroplast fraction would be approximately 20-25% of the total, roughly equivalent to what was observed in the sucrose density gradient intact chloroplast peak. However, quantitative determination of the estimates of in vivo enzyme content in the chloroplasts by a
oi
.6
0
w
August 1979
Fig.lA-C. Results of sucrose density gradient centrifugation of young, deribbed spinach leaves.
305
Volume 104, number 2
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August 1979
Table 1 Enzyme and chlorophyll distribution in sucrose density gradient fractions Component
Intact chloroplasts
Mitochondria and broken chloroplasts
Microbodies and soluble fraction
Chlorophyll &ml % of total Catalasea % of total Triosepho~hate isomerasea % of total Cytochrome oxidasea % of total p-Aspartate kinaseb % of total
1296.3 (48) 56.3 (9.91 3.92 (4.6) 0.411 (18) 4.44 (39)
894.2 (40) 81.3 (14) 4.41 (5.2) 1.586 (70) 3.04 (27)
140.1 (5.7) 409.2 (72) 65.66 (78) 0.084 (3.7) 3.22 (28)
a fimol/ml . mm b pmol/ml . h
calculation of recovered enzyme activity in relation to recovered chlorophyll in the intact chloroplast, used by Bryan et al. [3] for homoserine dehydrogenase, indicates that appro~m~ely 60% of the total &aspartate kinase activity is associated with the intact chloroplast . Analysis of the FPS and FPP fractions shows that the only detectable nitrite reductase activity was associated with the soluble fraction and that the
distribution of P-aspartate kinase is roughly equivalent in the two fractions; therefore, we do not yet know if &aspartate kinase is a soluble stromal or membranebound protein in the c~oropl~t. The data suggest that either two populations exist or fi-aspartate kinase sticks to the membrane. The intracellular distribution of fl-aspartate kinase in a leaf homogenate was also determined by another procedure, This involved centrifugation of a crude
Table 2 Distribution of nitrite reductase and @-aspartate kinase activities among differential centrifugation fractions Fraction
Crude Super, Super, Super 3 Pelletd 56 of pellet FPS FPP
CNorophyU
Protein
Nitrite reductase
@-Aspartate kinase
&ml
% total
mg/ml
% total
Total activitya
% total
Total activity b
% total
45 10 7.6 13 166
100 20 1.5 2.7 56
1.7 1.2 0.6 0.5 2.3
100 62 3 2.1 20
35.8 22.4 NDC ND 2.39
100 62 6.7
310 258 21.8 4.6 34.3
100 83 7.0 1.4 11
67 90
43 57
1.2 1.6
42 58
0.57 ND
26
a Units ‘=mmol NO; red/mm b Units = rrnoi AspHjmin ’ ND = not detectable dCharacteristics of chloroplasts -50% intact, fii 10 rmol CO,/mg chl * h
306
12.3 13.6
36 40
August 1979
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Volume 104, number 2
Table 3 Solubilization of p-aspartate kinase from isolated chloroplasts Specific activitya (units/mg protein)
+BSA (O.l%)b
Chlorophyll
-BSAb
% recovered
Total units
% total
% protein
Total units
% total
% protein
100
100
1142
100
100
832
70
71
5
13
Crude suspension Supernatant solution minus whole
2.17
100
2490
chloroplasts Extract from ruptured chloroplasts Supernatant/peUet enzyme ratio
1.85
42
1296
52
46
3.06
62
95
4
6
18.84
56.7 14.67
Under standard assay conditions, 60 min, 30°C, a and b are representative samples of separate experiments
homogenate at 500 X g for 1 min to sediment whole cells and debris. The resulting supernatant was centrifuged at 1500 X g for 5 min to sediment whole chloroplasts. Analysis of these fractions for /I-aspartate kinase activity yielded the following results (table 3). /3-Aspartatekinase specific activity in units/mg protein increased from 2.17 to 3.06 in the ruptured chloroplast fractions and was therefore enriched in the chloroplast pellet. The enrichment of the fl-aspartate kinase activity in the chloroplast pellet may be due to nonspecific binding of the protein to the membrane sites on the chloroplasts. To determine if P-aspartate kinase was binding nonspecifically, 0.1% bovine serum albumin was added to the extraction buffer, with the following results (table 3). /3-Aspartatekinase was approximately 5% of the crude preparation in both plus and minus bovine serum albumin preparations, showing no difference in the distribution of the enzyme activity. The supematant/pellet ratios were also approximately similar. The addition of bovine serum albumin to the extraction buffer also seemed to protect the enzyme activity, as noted in the increase in the total percentage recovered. Although some non-specific binding of /J-aspartate kinase may occur, the distribution of /.I-aspartate kinase in sucrose gradients and differential centrifugation indicates that there is a chloroplast-localized /3-aspartate kinase. Experiments are underway to characterize the chloroplast-localized P-aspartate kinase and the cytosolic /3-aspartate kinase, and to
determine if these are the same enzyme or isoenzyme forms by studying biochemical, kinetic and regulatory properties of the enzyme(s).
Acknowledgements This work was supported by the University of Tennessee Institute of Agriculture and US Department of Energy Contract no. EY-76-C-054242 with the University of Tennessee. The authors gratefully acknowledge the expert technical advice, critical review of the manuscript and use of equipment (oxygen monitor) provided by Dr Frederick J. Ryan and the critical review of the manuscript provided by Dr Otto J. Schwarz.
References [l] Lea, P. J., Mills, W. R. and Miflin, B. J. (1979) FEBS Lett. 98,165-168. [2] Mazehs, M., Miflin, B. J. and Pratt, H. M. (1976) FEBS Lett. 64,197-200. [3] Bryan, J. K., Lisik, E. A. and Matthews, B. F. (1977) Plant Physiol. 59,673-679. (41 Mills, W. R. and Wilson, K. G. (1978) FEBS Lett. 92, 129-132. [S] Miflin, B. J. and Beevers, H. (1974) Plant Physiol. 53, 870-874. [6] Itzaki, R. F. and Gill, D. M. (1964) Anal. Biochem. 9, 401-410. [7] Arnon, D. I. (1949) Plant Physiol. 24,1-15.
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[8] Schacterle, G. R. and Pollack, R. L. (1973) Anal. Biochem. 51,654-655. [9] Lilley, R. McC., Fitzgerald, M. P., Rienits, K. G. and Walker, D. A. (1975) New Phytol. 75, l-10. [lo] Walker, D. A. (1971) Methods Enzymol. 23,211-220. [ 1 l] Luck, H. (1965) in: Methods in Enzymatic Analysis (Bergmeyer, H.-U. ed) pp. 885-894, Academic Press, New York. [12] Hackett, D. P. (1964) in: Modern Methods of Plant Analysis (Linskins, H. F., Sanwal, B. D. and Tracey, M. V. eds) vol. VII, p. 647, Springer-Verlag. New York.
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[13] Gibbs, M. and Turner, J. F. (1964) in: Modem Methods of Plant Analysis (Linskins, H. F., Sanwal, B. D. and Tracey, M. V. eds) vol. VII, p. 520, Springer-Verlag, New York. (141 Losada, M. and Paneque, A. (1971) Methods Enzymol. 23,487-491. [15] Black, S. and Wright, N. G. (1955) J. Biol. Chem. 213, 27-38. [ 161 Henke, R. R. and Wahnbaeck, R. (1977) Biochem. Biophys. Res. Comm. 79,38-45. [ 171 Mifhn, B. J. (1974) Plant Physiol. 54,550-555.